Name: in vivo gene transfer to schwann cells in the rodent sciatic nerve by electroporation
Uploaded: 2016-09-08T15:00:00.000+00:00
Duration: 5 min 40 s
Description: Watch this Scientific Journal Video about In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation at JoVE.com

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology to M.I. (21229004 and 25221304).

L&#39;uso di topi per la ricerca era in conformità con le linee guida stabilite dal Comitato di benessere degli animali l&#39;Università di Tokyo. 1.Preparazione di plasmidi DNA <ol><li> Generare plasmidi DNA per elettroporazione in vivo subcloning cDNA o la sequenza shRNA in un plasmide di espressione per cellule di mammifero 12. Utilizzare un citomegalovirus immediato primi enhancer e pollo β-actina promotore di fusione (CAG) promotore-driven plasmide 13 perché permette l&#39;espressione forte e stabile. Per espressione shRNAs sotto il controllo di un promotore CAG, utilizzare un sistema a cassette shRNA mir30-based per la subcloning shRNA 14. </li><li> Purificare il DNA plasmide con un kit di maxi-prep secondo le istruzioni del fabbricante, e risospendere il DNA con soluzione salina HEPES-buffered (140 mM NaCl, 0,75 mm Na 2 HPO 4, 25 mM HEPES, pH 7,40). Regolare la concentrazione del DNA per ≥4 mg / mL. </li><li&gt; Preparare la soluzione plasmide DNA ad una concentrazione di 4 mg / mL, e aggiungere una quantità minima di colorante verde veloce (concentrazione finale di 0,01%) per etichettare il sito di iniezione. Quando è richiesto elettroporazione simultanea di più plasmidi, regolare la concentrazione totale della soluzione plasmide DNA a 4 mg / mL. Nota: La composizione ottimale dei DNA plasmidici deve essere determinata secondo la efficienza di trasfezione di ciascun plasmide. </li></ol> 2. sterilizzazione degli strumenti chirurgici e Saline <ol><li> strumenti chirurgici autoclave e la soluzione di NaCl allo 0,9%. </li></ol> 3. Preparazione della micropipetta di vetro <ol><li> Tirare pipette di vetro con un estrattore pipetta. Tagliare la punta della pipetta ad un diametro di 30-50 micron. Utilizzare i seguenti parametri: Calore, 600; Velocity, 50; Ora, 75. </li></ol> 4. Chirurgia Animale, iniezione del DNA ed elettroporazione Nota: Un overvista di questa fase è descritta in Figura 1. Sebbene la procedura di cuccioli di ratto sono descritti qui, il metodo è applicabile anche ad animali più maturi utilizzando la stessa procedura. <ol><li> Anestetizzare ratto con isoflurano nella casella di induzione finché l&#39;animale diventa immobile regolando il flusso di ossigeno al 0,4 L / min e la concentrazione isoflurano al 4% (vol / vol). Eseguire punta pizzicare per confermare il corretto anestesia. </li><li> Mettere il topo sul caldo caldo sotto un microscopio binoculare, e mantenere l&#39;anestesia con la somministrazione continua isoflurano attraverso la maschera. Regolare il flusso di ossigeno al 0,2 L / min e la concentrazione isoflurano al 2% (vol / vol). Utilizzare collirio per prevenire la secchezza degli occhi se gli occhi dell&#39;animale sono aperte. </li><li> Fissare le gambe con nastro chirurgico. </li><li> Pulire la pelle sulla coscia posteriore con iodopovidone, e fare un&#39;incisione con un bisturi. Nota: Rasatura aree chirurgiche se le aree chirurgiche sono coperte con haria. </li><li> Esporre il nervo sciatico, creando un varco tra le muscolo quadricipite femorale e del muscolo bicipite femorale con aghi da cucito. </li><li> Bagnare il nervo con 0,9% soluzione di NaCl. Assorbire l&#39;acqua in eccesso con carta privo di lanugine. </li><li> Inserire la base di una micropipetta di vetro sulla tubazione flessibile, e riempire la quantità adeguata di soluzione di DNA (almeno un microlitro) nella micropipetta aspirando delicatamente. </li><li> Sollevare il nervo scoperto tirando delicatamente la parte distale del nervo utilizzando un ago. Nota: Non applicare tensione al nervo per minimizzare lo stress meccanico. </li><li> Inserire la micropipetta di vetro nel sito distale sul nervo, ed iniettare la soluzione di DNA dalla pressione applicando (cioè soffiando nell&#39;estremità aperta del tubo flessibile). Iniettare soluzione di DNA fino a quando il nervo appare verde (massimo 1 ml). A causa frequente inserimento del micropipetta può danneggiare il nervo, non inserire la micropipetta più di due volte. </li><li> Posizionare un tweEzer-tipo di platino elettrodo circa 1-2 mm di distanza dal nervo. Colmare il divario tra l&#39;elettrodo e il nervo con 0,9% soluzione di NaCl. Nota: Non tenere il nervo con l&#39;elettrodo per evitare sollecitazioni meccaniche sul nervo. </li><li> Applicare impulsi elettrici al sito di iniezione utilizzando un elettroporatore con l&#39;elettrodo. Dopo il primo set impulso, invertire l&#39;elettrodo e applicare un altro set di impulso. Utilizzare i seguenti parametri: tensione, 50 V; durata dell&#39;impulso, 5 msec; Intervallo di impulso, 100 msec; numero di impulsi, 4 volte. </li><li> Pulire il sito di elettroporazione con il 0,9% soluzione di NaCl. </li><li> Ripetere i passaggi 4,4-4,11 sul nervo sciatico controlaterale. </li></ol> 5. Post-elettroporazione <ol><li> Chiudere le incisioni con la colla cianoacrilato. </li><li> Dopo l&#39;essiccazione della colla, pulire la ferita con povidone-iodio. </li><li> Rilasciare il cucciolo dalla maschera facciale. Riscaldare il cucciolo su un riscaldatore per almeno un&#39;ora in modo da consentire di recuperare completamente dall&#39;anestesia. Do non lasciare il cucciolo incustodito fino a quando non ha ripreso conoscenza sufficiente. </li><li> Dopo il recupero dall&#39;anestesia, restituire il cucciolo di ratto madre. Non restituire il cucciolo fino alla completa guarigione. </li></ol> 6. post-intervento chirurgico <ol><li> Casa i cuccioli di ratto in gabbia fino a condurre gli esperimenti 11 (si vedano gli esempi in figura 3). Somministrare carprofen (5 mg / kg, ip), un farmaco non steroideo anti-infiammatori, o buprenorfina (0,1 mg / kg, sc), un analgesico oppioide, se necessario. Nota: se il cucciolo di ratto non cresce bene o infiammazione si osserva attorno al sito chirurgico, escludere l&#39;animale dagli esperimenti. </li></ol>

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The authors declare that they have no competing financial interests.

In this paper, we describe a simple and efficient method that allows in vivo gene transfer to myelinating SCs in the rat sciatic nerve using electroporation. This method allows highly selective gene expression in myelinating SCs by simply applying electric pulses to the plasmid DNA-injected sciatic nerve. Because the molecular mechanisms of myelination and demyelination in the peripheral nervous system remain unclear, the present in vivo electroporation method will be a powerful tool to clarify the roles of multiple genes of interest in living animals.
A critical requirement of this method is to keep damage to the nerve during surgery to a minimal level. Should surgical damage cause excessive inflammation, the sciatic nerve may degenerate. To avoid this, one must conduct surgery with extreme care, so as to not damage the blood vessels around the nerve. Mechanical stress to the nerve during the surgery can also be a cause of nerve damage. To minimize mechanical stress, lifting the exposed nerve should be done as gently as possible, and the tweezer-type electrode should be placed close to the nerve without contact. Furthermore, electrical pulses that are too strong can cause undesirable large leg movement, which leads to mechanical stress, or can burn the nerve. If significant damages are observed in the nerves, we recommend reducing the electrical pulse intensities or placing the electrode further away from the nerve.
In our present protocol, CAG promoter-driven plasmids were used as expression vectors. CAG promoter-driven plasmids allow high levels of gene expression in myelinating SCs in vivo. We also have tried a CMV promoter, another widely used universal promoter for mammalian gene expression, but expression of the gene product was very weak. This is consistent with previous results, in which electroporation-mediated transfection was conducted in the embryonic brain20. Therefore, we recommend using CAG promoter-driven plasmids for the in vivo electroporation method.
Because axonal signaling is a key factor in myelination/demyelination21, gene modification in neurons is also important. However, delivery of transgenes using our in vivo electroporation method is limited to SCs. It has been reported that gene delivery into sciatic nerve axons can be achieved when in vivo electroporation is applied to dorsal root ganglion (DRG) neurons in adult rats22. This suggests that delivery of plasmid DNA to the cell body is likely to be critical for in vivo transfection of peripheral axons. Thus, to examine the involvement of axonal molecules in myelination/demyelination, researchers should use neuron-specific genetic methods such as genetically modified animals, neuron-specific viral vectors, or in vivo electroporation to DRG neurons.
Compared with current methods, such as the generation of genetically modified animal lines23 and delivery of transgenes by viral vectors4-6, gene modification of SCs by in vivo electroporation is simpler. This method only requires several days for plasmid DNA construction and one day for electroporation surgery. Plasmid DNA construction does not require a biohazard room that is usually essential for viral vector handling. In addition, one of the advantages of the electroporation method is the capacity for simultaneous expression of multiple gene products using a simple protocol. Our novel technique will be useful for analyzing the interaction of a variety of signaling molecules involved in myelination and demyelination. In particular, by permitting the cotransfection of a number of different intracellular fluorescent probes, our method should be a powerful tool for investigating intracellular signaling dynamics in SCs using live imaging experiments.

The rapid transmission of sensory and motor information in the peripheral nervous system is permitted by the myelin sheath, which is formed by myelinating Schwann cells (SCs)1. Insulation of axons by the myelin sheath enables saltatory conduction, which increases the speed of nerve impulses. In disorders in which the development or maintenance of the myelin sheath is impaired, nerve conduction speed is reduced. This results in neuropathy involving motor and sensory dysfunction. Although there are many studies on the molecular mechanisms of myelination and demyelination in the peripheral nervous system, the roles of the numerous proteins involved in these processes remain unclear.
To study the molecular mechanisms of SC myelination/demyelination in vivo, genetic approaches have been used to modify gene expression in animals. A powerful approach is the use of knockout/knockin or transgenic animals. However, the generation of these animals is expensive and time consuming. For SC-specific gene manipulation, crossing floxed strains with Cre mice or other conditional gene expression methods are necessary. This again is laborious and time intensive. In recent years, a cutting-edge genetic technology, the CRISPR-Cas9 system, has made the generation of genetically modified mice much quicker (about 4 weeks)2,3, but this method is hindered by target sequence limitations, and suffers from off-target effects. As an alternative method, viral vector-mediated gene transfer is a faster and easier method of achieving gene transfer into SCs in vivo4-6. Indeed, the generation of viral vectors is less expensive, and takes a shorter time (within a few weeks), and gene manipulation of SCs can be achieved by simply injecting engineered viral vectors, such as adenoviral vectors, adeno-associated viral (AAV) vectors, and lentiviral vectors, into the sciatic nerve. Because these viral vectors have different characteristics, users have to choose the one best suited for their purpose. Adenoviral vectors infect axons and SCs in both young and mature sciatic nerves. In particular, adenoviral vectors have higher selectively for non-myelinating SCs than myelinating SCs. Adenoviruses can cause immune responses, and accordingly, immunodeficient strains should be used5. AAV vectors are currently the most widely used viral vectors, and allow in vivo gene transfer with lower toxicity7. AAV can transduce both axons and SCs by direct injection into the nerve fibers8,9. However, AAV-mediated protein expression usually requires 3 weeks or longer to reach maximum levels7,9. Therefore, it is difficult to analyze myelination, which actively progresses during the two week postnatal period. Lentiviral vectors have higher selectively for myelinating SCs than non-myelinating SCs, and do not have toxic effects on sciatic nerves. However, lentiviral vectors do not infect SCs in more mature nerves5, and therefore are unsuitable for analyzing events such as the demyelination process.
Electroporation is another faster and easier approach to achieve in vivo gene transfer. It has been reported that in vivo transfection of SCs can be achieved when electroporation is applied to transected rat sciatic nerves10. However, because this method requires nerve transection for gene delivery, the application is limited to the analysis of the damaged nerves. Here, we describe an alternative method that allows the delivery of transgenes into myelinating SCs in intact rat sciatic nerves using electroporation11. This method requires plasmid construction, which can usually be completed within a week. Then, by simply delivering electric pulses to the site on the sciatic nerve where the plasmid DNA was injected, highly selective transfection of myelinating SCs can be achieved in neonatal as well as in more mature animals. By electroporating multiple plasmids, simultaneous expression of a variety of genes can be easily achieved. The ability to simultaneous express multiple molecules, such as signaling proteins, short-hairpin RNAs (shRNAs) and functional probes, is crucial for investigating complex processes such as myelination and demyelination. The novel in vivo electroporation method described in this paper will be a powerful tool, allowing researchers to analyze the function of a multitude of molecules and their interactions in myelinating SCs.

<table><tbody><tr><td>Genopure Plasmid Maxi Kit</td><td>Roche</td><td>03 143 422 001</td><td>Plasmid DNA purification kit</td></tr><tr><td>Fast Green CFC</td><td>WAKO</td><td>069-00032</td><td>Dye for DNA injection</td></tr><tr><td>GC 150T-10</td><td>HARVARD APPARATUS</td><td>30-0062</td><td>Glass capillary</td></tr><tr><td>Suction tubing</td><td>Drummond</td><td>05-2000-00</td><td>Suction tubing for micro injection</td></tr><tr><td>MODEL P-97</td><td>SUTTER INSTRUMENT CO.</td><td>Micropipette puller</td><td></td></tr><tr><td>CUY21 Single Cell</td><td>BEX</td><td>Electroporator CUY21 Single Cell</td><td>Pulse generator</td></tr><tr><td>Electric warmer</td><td>KODEN</td><td>CAH-6A</td><td>Warmer during the surgery</td></tr><tr><td>Isofluolane</td><td>Mylan</td><td>1119701G1076</td><td>Anesthetic</td></tr></tbody></table>

in vivo gene transfer to schwann cells in the rodent sciatic nerve by electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

Un esempio di un nervo sciatico transfettate con la proteina fluorescente rossa (RFP) esprimente il plasmide è mostrato in Figura 2A. Le cellule che mostrano la morfologia bipolare, una caratteristica della SCS, sono stati scarsamente trasfettate con RFP. No RFP fluorescenza è stato rilevato negli assoni. Noi di solito troviamo ~ 100 SC trasfettate in ogni nervo. Questa efficienza trasfezione sembra simile alla SC efficienza in vivo infezione utilizzando vettori lentivirali 4. Esperimenti immunostaining hanno dimostrato che la maggior parte (~ 96%) cellule RFP-positive a P7 co-marcato per S100, un marcatore SC (Figura 2B) e 91% di cellule RFP-positive a P14 co-marcato per MBP, un marcatore myelinating SC (Figura 2C), suggerendo che il trasferimento genico mediante elettroporazione è altamente selettivo per mielinizzanti SC. L&#39;introduzione di geni multipli in SC <em&gt; in vivo sarà estremamente utile per studiare i meccanismi di mielinizzazione / demielinizzazione. Uno dei principali vantaggi del metodo nel vivo elettroporazione qui descritto è la capacità di trasferire geni multipli con una procedura semplice. La figura 2D mostra un&#39;immagine rappresentativa di un nervo sciatico trasfettate con una miscela di GFP e RFP plasmidi che esprimono l&#39;utilizzo nel elettroporazione in vivo. Circa il 97% di SC erano GFP e RFP doppio positive, suggerendo che la consegna altamente efficiente di geni multipli può essere ottenuto semplicemente electroporating miscele di diversi plasmidi. Nei roditori, mielinizzazione inizia intorno nascita, aumentando notevolmente durante le prime due settimane dopo la nascita, e quindi diminuisce gradualmente. Così, manipolando geneticamente SC durante queste finestre temporali di sviluppo, i meccanismi alla base di queste diverse fasi di mielinizzazione possono essere chiarite. vettori lentivirali sono un ottimo strumento per unnalyzing mielinizzazione, in particolare per quanto hanno tossicità minima, ma lentivirus di infettare solo neonatale nervi sciatico 5,6. In confronto, il trasferimento genico elettroporazione mediata funziona bene quando trasfezione viene condotta su P3 (figura 2E, in alto) o P14 (Figura 2E, bottom). Le applicazioni del romanzo nel metodo di elettroporazione in vivo sono descritte qui. La figura 3A mostra le immagini microscopiche luce di GFP che esprimono SC mielinizzanti a vari stadi di sviluppo (P7, P14, P21 e P31). Con analisi al microscopio ottico, alterazioni dei parametri morfologici, come la lunghezza e il diametro, possono essere valutate. Si noti che questi parametri hanno valori simili rispetto al intatto ratto nervi periferici 15,16, il che suggerisce che i nervi elettroporate si sviluppano senza rilevanti conseguenze dannose. La figura 3B mostra un elettrone un&#39;immagine microscopica della LacZ-espresssing mielinizzanti SC. In questo caso, LacZ è stato utilizzato come indicatore di espressione. β-galattosidasi colorazione utilizzando bluO-gal, un substrato di etanolo insolubile, consente l&#39;analisi della struttura della mielina del SC trasfettate al microscopio elettronico 11,17. In questi esperimenti, il ruolo delle molecole di segnalazione può essere esaminato da tacere o aumentando la loro espressione, consentendo in tal modo l&#39;analisi di perdita-di-funzione o il guadagno-di-funzione effetti. Oltre all&#39;analisi di tessuto fissato, in vivo trasferimento genico elettroporazione mediata può essere applicato anche a vivere esperimenti di imaging. Ad esempio, la Figura 3C mostra una myelinating SC co-esprimono G-GECO1.1 18, un indicatore verde fluorescente citosolico Ca 2+, e R-GECO1mt 19, un indicatore rosso fluorescente mitocondriale Ca 2+. Esprimendo questi indicatori, abbiamo identificato un percorso di segnalazione che controlla citosoliche e mitocondriali Ca 2+ concentrazioni in mielinizzanti SC<html> . Pertanto, il presente metodo può essere usato per studiare una varietà di meccanismi di segnalazione, in particolare quando le sonde fluorescenti geneticamente codificati sono disponibili per rilevare i segnali di interesse. <img alt="Figura 1" src="/files/ftp_upload/54567/54567fig1.jpg" /> Figura 1:. Schematica della i n elettroporazione in vivo Metodo Innanzitutto, il nervo sciatico del ratto anestetizzato è esposto. In secondo luogo, il plasmide DNA viene iniettato nel nervo sciatico. In terzo luogo, gli impulsi elettrici vengono consegnati al sito di iniezione attraverso l&#39;elettrodo a forma di pinza. Infine, la ferita viene chiusa con colla. Questa procedura può essere ripetuta sul nervo controlaterale. <a href="https://www.jove.com/files/ftp_upload/54567/54567fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> &quot;&gt; <img alt="figura 2" src="/files/ftp_upload/54567/54567fig2.jpg" /> Figura 2: Risultati Rappresentante sui nervi sciatico trasfettate (A) Una immagine rappresentativa di un nervo sciatico transfettate.. Il nervo è stato transfettate con plasmide RFP che esprimono al P3, e fissato a P7. (B) Una immagine rappresentativa di una cellula RFP-transfettate a P7 mostrando colocalizzazione con S100, un marker SC. (C) Una immagine rappresentativa di un nervo sciatico RFP-trasfettate a P14 che mostra colocalizzazione con MBP, un marker myelinating SC. (D) Una immagine rappresentativa di un nervo sciatico cotransfected con GFP e RFP plasmidi che esprimono. SC trasfettate espresso contemporaneamente GFP e RFP. (E) Un&#39;immagine di mielinizzanti SC a P31 trasfettate al P3, quando inizia la mielinizzazione (in alto), e un&#39;immagine della SCS mielinizzanti a P31 transfettate a P14, quando la maggior parte di grandi assoni diventano mielinato (in basso), suggerendo che trasfezione di SC mielinizzanti può essere raggiunto non solo nei nervi neonatale, ma anche nei nervi più maturi. Barre di scala = 200 micron (A); 50 micron (BE). Questa cifra è stata modificata dalla nostra precedente pubblicazione 11. <a href="https://www.jove.com/files/ftp_upload/54567/54567fig2large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54567/54567fig3.jpg" /> Figura 3: Applicazione di elettroporazione in vivo (A) L&#39;analisi al microscopio luce dello sviluppo di mielinizzanti SC.. nervo sciatico sono stati elettroporate con plasmide GFP che esprimono al P3, e sono stati fissati a vari stadi di sviluppo (P7, P14, P21 e P31). Immagini rappresentative della SC GFP-positive sono mostrati sulla sinistra. La lunghezza media e diametro sono riassunti come media ± SEM (n = 30-47 da 3 nervi) sulla destra. La lunghezza e il diametro mielinizzanti SCs aumenta al procedere dello sviluppo. (B) Un elettrone immagine al microscopio di un nervo sciatico trasfettate con un plasmide codificante LacZ. A transfettate SC (asterisco bianco, sinistra) è stato finemente etichettato con precipitati del prodotto di reazione β-galattosidasi. (C) L&#39;immagine di una SC cotransfected con G-GECO1.1, un indicatore verde fluorescente citosolico Ca 2+, e R-GECO1mt, un indicatore rosso fluorescente mitocondriale Ca 2+. Le regioni all&#39;interno dei bianchi rettangoli tratteggiate sono mostrati ampliata nei pannelli a destra. Barre di scala = 50 micron (A e C); 1 micron (B). Questa cifra è stata modificata dalla nostra precedente pubblicazione 11. <a href="https://www.jove.com/files/ftp_upload/54567/54567fig3large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation at JoVE.com

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In Vivo Gene Transfer di cellule di Schwann nel nervo sciatico roditori da elettroporazione

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

in-vivo-gene-transfer-to-schwann-cells-rodent-sciatic-nerve

Nanyang Technological University

Research

JoVE Journal

Neuroscience

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

8.9K Views.  The University of Tokyo. Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Video: In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Gene consegna di cervello di ratto post-natale da parte di non-ventricolare iniezione plasmidi e Elettroporazione

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Ricerca

Neuroscienze

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Gene Transfer per l&#39;orecchio del mouse Sviluppo Inner<em&gt; In Vivo</em&gt; Elettroporazione

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

In Misure Vivo elettrofisiologici sui nervi mouse sciatico

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Sottotipo-selettivo elettroporazione della corticale interneuroni

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

Trapianto di Schwann Cells PVDF-TrFE condotto a ponte ratto Transected del midollo spinale ceppi per promuovere la rigenerazione degli assoni attraverso il Gap

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...

Medicina

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Inquirenti rigenerazione dell'assone dei mammiferi: In Vivo elettroporazione del ganglio di radice dorsale del topo adulto

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

In Vivo trasfezione di siRNA e Gene Knockdown in Spinal Cord via rapida non invasiva lombare spinale iniezioni nei topi

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Biologia

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

Mielinizzazione in vitro di assoni periferici in una cocoltura di espianti di ganglio di radice dorsale di ratto e cellule di Schwann

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...